With 8.9 million new cases and 1.7 million deaths per year tuberculosis is a leading AIDS-related global killer which has not been effectively controlled. The causative agent, Mycobacterium tuberculosis, proliferates within host macrophages where it modifies both its intracellular and local tissue environment resulting in caseous granulomas with incomplete bacterial sterilization. We recently reported that M. tuberculosis uses at least one of its adenylate cyclases (Rv0386) to secrete cAMP into the host cell cytoplasm. By this cAMP intoxication mechanism, M. tuberculosis subverts host cell signal transduction by induction of protein kinase A (PKA), upregulation of cAMP-response element binding protein (CREB) phosphorylation, and a cascade of additional responses including excess TNF-1 secretion. Recently we have begun to probe other consequences of cAMP intoxication using novel recombinant M. tuberculosis strains with altered ability to deliver cAMP, through microarray and multiplex cytokine profiling, and through pharmacologic inhibition of key cAMP signaling pathways. We have found that cAMP intoxication may block induction of the key host cell anti- microbial peptides (AMPs) cathelicidin and human 2-defensin-1. Also, we found that pharmacologic inhibitors of phosphodiesterases modulate the outcome of murine tuberculosis possibly by interfering with the cAMP intoxication pathway. In Aim 1 of this study we will systematically address the consequences of cAMP intoxication at the cellular level in vitro, in whole lung tissue using the mouse model in vivo, and at the granuloma level using laser-capture microdissection in specialized animal models including the rabbit which display caseation necrosis. In Aim 2 we will investigate the ability of clinically available signal transduction inhibitors (PDE inhibitors, JAK inhibitors) to modulate the cAMP intoxication pathways identified in Aim 1, and we will test some of these drugs for their ability to accelerate cure when used as adjuvant immunomodulatory drugs in the TB mouse model. In Aim 3 we will investigate whether bacterial mechanisms to elevate intramacrophage cAMP levels may inhibit the expression of host AMPs (cathelicidin and human 2-defensin-1) and determine the extent to which vitamin D induction of AMPs may override this mycobacterial virulence mechanism. PUBLIC HEALTH RELEVANCE: The bacteria causing TB enter human cells and subvert normal human cell signaling by secreting excess levels of the second-messenger, cyclic-AMP;we have observed that this process enables the microbe to block host cell secretion of key defense molecules known as anti-microbial peptides. This study will identify the specific mechanisms and inflammatory processes that are subverted by bacterial cyclic-AMP secretion and will test clinically available drugs which may interfere with the microbial cAMP-mediated subversion.